This research proposal focuses on the role of FAM20C (also known as dentin matrix protein 4 or DMP4) in the phosphorylation of SIBLING proteins. Our recent data have established that FAM20C is essential to the formation and mineralization of bone and tooth, and that this protein kinase may be the primary enzyme responsible for catalyzing the phosphorylation of SIBLING proteins. We discovered: 1) FAM20C is highly expressed in the cells forming bone, dentin, cementum and enamel; 2) the inactivation of Fam20C in mice leads to rickets with extensive hypomineralization in murine bone; 3) Fam20C-KO mice have significant defects in the formation and mineralization of dentin, cementum and enamel; 4) the total phosphorylation level of non-collagenous proteins in the bone or dentin of Fam20C-KO mice is significantly lower than that of wild type mice. Furthermore, we and others revealed that recombinant FAM20C effectively phosphorylates some of the SIBLING proteins in vitro. Our preliminary data lead us to form the central hypothesis that FAM20C is the primary kinase catalyzing the phosphorylation of SIBLING proteins and the defects in the hard tissues of the Fam20C-KO mice may result from a significant loss of phosphorylation in the SIBLING proteins. To test this novel hypothesis, we propose the following three specific aims: (1) To determine the effects of FAM20C deficiency on the phosphorylation of SIBLING proteins. We will isolate OPN, BSP, the C-terminal fragment of DMP1, and DPP from the bone and dentin of Fam20C-KO mice and compare the phosphorylation level of these proteins with that in tissues from WT mice. We will also examine the effects of FAM20C on the phosphorylation of SIBLING proteins via 32P incorporation assay in the cultures of calvarial osteoblasts derived from Fam20C-KO and WT mice. (2) To examine phosphoserines in the SIBLING proteins isolated from the bone and dentin of Fam20C-KO and WT mice by digesting OPN, BSP, DMP1 C-terminal fragment, and DPP with proteases, separating the proteolytic peptides, and sequencing the isolated peptides with mass spectrometry. (3) To examine the effects of SIBLING proteins isolated from the Fam20C-KO mice on the mineralized nodule formation by osteoblasts and odontoblasts. The multipronged approaches proposed in this application will advance our understanding of the role of FAM20C in the phosphorylation of SIBLING proteins, and serve as the foundation for further elucidating the mechanism of biomineralization associated with the phosphorylation of SIBLINGs.